A natural mutation in the promoter of Ms-cd1 causes dominant male sterility in Brassica oleracea

Male sterility has been used for crop hybrid breeding for a long time. It has contributed greatly to crop yield increase. However, the genetic basis of male sterility has not been fully elucidated. Here, we report map-based cloning of the cabbage (Brassica oleracea) dominant male-sterile gene Ms-cd1 and reveal that it encodes a PHD-finger motif transcription factor. A natural allele Ms-cd1PΔ−597, resulting from a 1-bp deletion in the promoter, confers dominant genic male sterility (DGMS), whereas loss-of-function ms-cd1 mutant shows recessive male sterility. We also show that the ethylene response factor BoERF1L represses the expression of Ms-cd1 by directly binding to its promoter; however, the 1-bp deletion in Ms-cd1PΔ−597 affects the binding. Furthermore, ectopic expression of Ms-cd1PΔ−597 confers DGMS in both dicotyledonous and monocotyledonous plant species. We thus propose that the DGMS system could be useful for breeding hybrids of multiple crop species.

ZmMs7 in maize.MS1 is extensively studied during anther development.Its knockdown/out leads to TGMS phenotype in rice and male sterile in all plants investigated.However, a 1-bp deletion in the transcriptional regulatory region of the promoter of Ms-cd1 leads to overexpression and dominant genic male sterility.Furthermore, an ethylene response factor (ERF) BoERF1L was found to directly bind to the promoter of Ms-cd1PWT to repress Ms-cd1 expression.The genetic data is very solid in this work which clearly demonstrates the mechanism of DGMS phenotype.
Although both overexpression and knockout of Ms-cd1 leads to male sterile, the molecular and cellular mechanisms are quite different.I would like to know how overexpression of this gene leads to male sterile.The ectopic expression of Ms-cd1P-597 conferred DGMS to both dicotyledonous and monocotyledonous plant species, including rice, rabidopsis, tomato, and rapeseed.In Arabidopsis, the genetic pathway DYT1-TDF1-AMS-MS188-MS1 is critical for tapetum development and pollen formation.The expression analysis of this pathway and their downstream genes in P-597::Ms-cd1 (Arabidopsis) plant may provide clues of the DGMS mechanism.
Reviewer #3: Remarks to the Author: Fengqing Han et al. reported the map-based cloning of the dominant male-sterile gene Ms-cd in cabbage, which encodes a transcription factor homologous to MS1 in Arabidopsis, PTC1/OsMS1 in rice, and ZmMs7 in maize.Ethylene response factor (ERF) BoERF1L directly bound to the GAC-core motif in the promoter and repressed the expression of Ms-cd.The 1-bp deletion in the Ms-cd1P-597 prevented this binding ability and enhanced the promoter activity, leading to severe pollen wall defects and conferring dominant genic male sterility (DGMS) to Brassica oleracea.The authors found that a rare 1 bp deletion in the Ms cd1 promoter causes male sterility, but more data is needed to support their conclusions.
The authors observed that P 597 has higher promoter activity, and concluded that the expression level, but not the tissue-specific expression pattern, of Ms cd1 is altered in DGMS plants.I have not found data to support this conclusion.
Both knocking out and enhancing Ms-cd1 leads to male sterility, the mechanism remains unknown.This point can be discussed.
The dual LUC assay showed that the BoERF1L repressed PWT::LUC reporter transcription, but more evidence is expected to show that BoERF1L regulates Ms cd1 expression.
Please show evidence that RNAi 4 and RNAi 6 are RNAi transgenic plants.
BoERF1L as an ERF transcription factor should respond to plant hormones and pathogen attacks and abiotic stresses.So male sterility/fertility will be sensitive to environmental stress.
In the result section lines 278-279: A promising DGMS system for heterosis utilization in dicotyledonous and monocotyledonous crop species.In the example shown in Figure 6E, they didn't show the data of heterosis.I also didn't see the results in monocotyledonous crop species.

Point-by-point responses
Reviewer #1 (Remarks to the Author): The manuscript entitled "A rare 1-bp deletion in the promoter of Ms-cd1 causes dominant male sterility in 2 Brassica oleracea" by Han et al. described the cloning of a dominant male sterile gene, which encodes a transcription factor.Its orthologs in several other species have been shown to be associated with pollen development.Their loss-of-functions led to male sterile in a recessive manner.Unlike the male sterile in other species, the male sterile investigated by Han et al. is a dominant one, caused by a 1-bp deletion in its promoter region.The authors showed that the 1-bp deletion was disabled the binding of a repressor transcription factor ERF1L.The 1-bp promoter deletion consistently led to a strong promoter in several species including tomato and rice.If I understand it correctly, the results suggest that the binding motif of ERF1L and its repression on Ms-cd1 (PHD-finger) are conserved in different plant species.The manuscript is well prepared, easy to read.
The results are sound, provide new insights on dominant male sterile, and can be applied in breeding programs in Brassica oleracea and potentially in other crops as well.